Acta anat. 102: 94-101 (1978)
Histocheniical studies on the peroxidase localization in the rat ovary and uterus during various reproductive stages Prabha Agrawal and Mannwhan M . Laloraya Department of Life Sciences, University of Indore, Indore, India
Key words. Ovary • Rat • Peroxidase • Reproductive cycle • Pregnancy
Introduction The different components of the ovary, i.e. the follicle, corpus luteum and stroma, undergo complex morphological, histocheniical and biochemical changes during the reproductive cycle of the rat. After ovulation, the cells of the membrana granulosa show hypertrophy and become luteal cells which secrete massive amounts of progesterone. Progesterone is known to be a pre cursor of several other steroid hormones including estradiol that controls folliculogenesis. The corpora lutea of many mammals are the chief site of conversion of “ C-acetate to progesterone [Zander el at.. 1959; Hammerstein el a!., 1964; Savard el id.. 1965] and are known to be characterized by the presence of large quantities of sudanophilic lipids and the presence of the specific steroid dehydrogenases |Levy el al., 1959; Guraya. 1974], Steroidogenesis in the reproductive target organs has been related to the functioning of large number of specific dehydrogenases and isomerases [Villee and Engel. 1961], In the presence of specific dehydrogenases,
operative both during the development of the follicle in the membrana granulosa [Deane el al.. 1962; Rubin el al., 1963; Label and Levy, 1968] and in the derived corpus luteum, how the rate of oxidation reaction changes in such a manner so as to stimulate synthesis of progesterone at a faster rate is largely unknown. Recently, luteinizing hormone has been shown to in duce peroxidase in the corpora lutea accompanying the depletion of ascorbate in the rat ovary, and suggestion has been made that the free radical of ascorbate produced by the action of peroxidase on ascorbic acid may trigger oxidation of pregnenolone through a free radical mechanism, thus bringing about rapid forma tion of progesterone [Agrawal and Laloraya. 1977], The presence of peroxidase may, therefore, serve as a useful histocheniical indication of the process of lutcinization associated with intense vascularization of the ovary that occurs in response to luteinizing hormone. The present work reveals that not only the corpora lutea but also the uterus of the pregnant rats and the associated placenta, known to secrete large quantities of pro gesterone during pregnancy, are characterized by very high localization of peroxidase activity.
Downloaded by: King's College London 137.73.144.138 - 11/21/2017 6:43:11 PM
Abstract. A histocheniical study has been made of peroxidase changes in rat ovary and uterus during different developmental stages of the normal and pregnant rats. The peroxidase was found to be present in the corpus luteum of the ovary of both normal and pregnant rats as well as in the allantochorionic placenta, while the growing follicles in the immature and mature rat ovary showed no activity. The possible physiological significance of the peroxidase changes in relation to luteal steroidogenesis has been discussed.
Agra wal / Laloraya
95
Materials and methods
Observations The histochemical features of the ovary and uterus at different stages in normal and preg nant rats, especially in relation to the ocurrence of peroxidase and steroidogenesis, will be described here separately. Immature rat I-month-old rats show compact stroma tissue full of growing follicles and interstitial gland tissues (IGT). The granulosa cells of normal growing follicles are small. No activity of peroxidase could be seen either in the mem brana granulosa of the growing follicles or in the IGT (fig. I). The granular dark deposits seen in the stroma region and membrana gra nulosa are due to yellowish brown lipid drop lets which do not show peroxidase activity.
Fig. 1. A portion of a I-month-old rat ovary show ing no localization in the membrana granulosa (MG) and IGT.
Mature rat With the appearance of antrum filled with liquor folliculi, the granulosa and the sur rounding stromal elements begin to hyper trophy. In the final stages of follicular growth, the granulosa cells form a relatively thin layer, called membrana granulosa. The theca interna of the growing follicle consists of stromal ele ments which do not show much cytoplasmic differentiation. No peroxidase activity could be seen in the membrana granulosa while the theca interna cells of the atretic follicles show high peroxidase activity (fig. 4). As compared with the original granulosa cells, the luteal cells are hypertrophied glan dular tissue which is well vascularized. The most striking and significant change during the transformation of granulosa cells is the ap pearance of high peroxidase activity through out the cytoplasm of the latter. Ovaries from mature rats show heavily stained corpus
Downloaded by: King's College London 137.73.144.138 - 11/21/2017 6:43:11 PM
Colony-bred albino rats of different age groups of Wistar strain, maintained under laboratory conditions, were used in the present study. Mature rats showing regular 4- or 5-day cstrus cycles were sacrificed at estrus phase of the cycle which was checked by pre paring vaginal smears. Pregnancy, which lasted 21 days, was induced by mating with proven males, and only confirmed pregnant rats were taken for the study. Peroxidase was localized in freshly cut frozen sections (8-10 nm) of the ovary and uterus by the method of Van Dnija [1951] using benzidine as donor. The activity was also tested with two other donors, namely guaiacol and p-anisidine. The presence of peroxidase was indicated by the specifically colored products of the reaction: benzidine producing deep navy blue turning to dark brown, p-anisidine pink and guaiacol brown products. The specificity of the per oxidase system was checked by keeping controls with out H20 2, i.e. with donors alone, and by treating the sections for 30 min with different concentrations of KCN, namely 10 2 10 M potassium cyanide, before performing the lest.
Fig.2. A portion of preovulatory ovary of rat showing no activity of peroxidase in the growing follicles (GF). The membrana granulosa (MG) of a large growing follicle shows no activity of peroxidase, while the dense patches of IGT with no peroxidase activity are seen.
Fig.3. A portion of mature rat ovary showing localization of peroxidase in the corpus luteum (CL), while no activity could be seen in the membrana granulosa (MG) of the growing follicle.
Agrawal/Laloraya
Fig.4. A portion of a postovulatory 4-month-old rat ovary showing localization in the theca interna (TI) of atretic follicles and dotted localization (DL) in the stromal region. The black area seen in the IGT is due to heavy deposition of lipid material which does not give any reaction with any of the three donors in the peroxidase system.
Fig.5. A high-power view of corpus luteum (CL) from the mature ovary showing dense deposition of peroxidase in the corpus luteum.
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Fig. 6, 7. A portion o f the rat ovary at estrus showing intense navy blue localization of peroxidase in the corpora lutea (CL) and dotted localization (DL) in the stromal region. The IGT is seen with yellowbrown fat deposits showing no peroxidase activity. Fig. 8. A high-power view of corpus luteuni (CL) from the rat ovary at estrus showing dense navy blue deposition of peroxidase in the luteal cell mass (CL). No activity is seen in IGT containing yellow-brown fat deposits.
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Peroxidase changes during the reproductive cycle of rat
Fig. 9. A portion of postovulatory ovary showing diffuse localization of peroxidase in corpus albicans (CA).
Fig. 10. Cyanide-treated section of corpus luteum (CL) showing complete inhibition of peroxidase locali zation. Dotted localization (DL) is not affected by cyanide which is seen in the stromal region (S).
Agrawal/Laloraya
luteum (fig. 5, 6) with no activity in the mem brana granulosa of the growing follicles and IGT (fig. 2, 3, 6, 7). However, corpora lútea in ovary exhibited different intensities of per oxidase activity which appears to change with the regression of corpus luteum (fig. 9). The black patches observed in the IGT are again due to massive accumulation of yellowbrown lipid droplets devoid of any peroxidase activity. No activity was observed without H.,02 in the system and cyanide exerted a powerful inhibitory effect (fig. 10). The stromal elements, which are apparently derived from the theca interna and theca ex terna, are of small size. Apart from heavy localization in the corpus luteum, the stroma region shows sparsely distributed dotted cel lular localization (fig. 6). Cyanide treatment almost completely inhibited the localization in the corpus luteum. but the scattered dotted cellular localization was not much affected. Also the scattered dotted cellular localization was observed with another donor, namely, guaiacol. No localization could be obtained with p-anisidine as donor. These observations raise doubt about the presence of peroxidase activity in these cells. No activity of the per oxidase was seen in the uterus of the normal mature rat (fig. 12). The myometrium and se rous coat of the uterus show dotted cellular localization which was not inhibited by cya nide. The dotted localization observed in the stroma tissue of the ovary, in the myometrium and serous coat of the uterus, which is in sensitive to cyanide, appears to be due to the presence of some oxidizing substances which are known to produce the colored oxidation products nonenzymically. Pregnant rat During early pregnancy, well-vascularized luteal cells present in the ovary showed intense
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Fig. 11. Part of rat corpus lutcum (CL) in early stages of pregnancy showing intense activity of per oxidase in the luteal cell mass. Stroma (S) shows dotted localization.
Fig. 12. Uterus of normal rat showing only dotted localization (DL) in myometrium (M) and serous coat (SC). Note that no localization is seen in the endo metrium (H).
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Fig. 13. Uterus of pregnant rat showing heavy localization of peroxidase in epithelial cells of the endometrium (E) while dotted localization (DL) is seen in the stromal region (S). The myometrium (MM) shows no activity.
Fig. 14. A portion of pregnant rat uterus from the early half of gestation, showing navy blue deposition of peroxidase in the endometrium (E) while dotted localization is seen in the stroma (S) region. The myo metrium (M) shows no activity.
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Peroxidase changes during the reproductive cycle o f rat
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Agrawal/Laloraya
Thus, the growing follicles in the immature as well as the mature rats showed no perox idase activity, while the corpora lutea in the mature rats and of the pregnant as well as the allantochorionic placenta showed intense per oxidase activity.
Fig. 15. A high-powcr view of early embryo (EY) in the uterus. The allantochorionic placenta (ACP) shows intense activity of peroxidase. The black patches ob served in the embryo a re. due to its compact nature and yellow-brown color which was devoid of peroxidase activity.
peroxidase activity (fig. 8, 11 ). The intensity of the localization was much higher as compared to the corpora lutea of normal rats at estrus. The uteri of pregnant rats show a welldeveloped decidua exhibiting extremely high peroxidase activity (fig. 13, 14). No activity could be seen in the myometrium and serous coat of the uterus at this stage of pregnancy. The entire length of the decidua was deeply stained, the endometrium lining exhibiting maximum localization. Some dotted cellular localization is seen in the stroma region of the decidua. The attachment between the embryo and the decidua, i.e. allantochorionic placenta, which develops during the later stages of preg nancy, showed intense localization of per oxidase (fig. 15).
The presence of peroxidase in the spleen, stomach, adrenal glands, eosinophils, intestine and liver has been reported [Saunders et at., 1964], Lucas et at. [1955] have shown the pres ence of peroxidase in the uterus while it under goes rapid proliferation during the different phases of the cycle, and this activity is even more pronounced in the estrogen-stimulated uterus of the ovariectomized rat. These reports of the occurrence of peroxidase in the genital organs, however, did not specify the localiza tion and its possible role in luteal steroidogen esis. Guraya [1974], on the basis of presence of sudanophilic granules and its changes under gonadotrophic hormone action, has suggested the possibility of interstitial gland cells acting as sites for steroidogenesis. These sites are well known to be associated with storage of lipid material including phospholipids and triglycerides. The observation that cholesterol could be demonstrated in interstitial gland cells and under conditions of lowered progesterone secretion and not during active secretion might suggest that the mobilization of the precursors for the biosynthesis of progesterone is con trolled by the corpus luteum. Since no activity of peroxidase could be demonstrated in inter stitial gland cells it appears to be an unlikely site for luteal steroidogenesis. The corpora lutea of many mammals are the chief site of conversion of "C-acetate to
Downloaded by: King's College London 137.73.144.138 - 11/21/2017 6:43:11 PM
Discussion
Peroxidase changes during ihe reproductive cycle of rat
References Agrawal, P. and Laloraya, M.M.: Induction of per oxidase in corpora lutea of rat ovary by luteinizing hormone. Biochem. J. 166: 205 208 (1977) Deane. H .W .; Lobel, B.L., and Romney, S. L.: En zymic histochemistry of normal human ovaries of the menstrual cycle, pregnancy and the early puerperium. Am. J. Obstet. Gynec. 83: 281-294 (1962). Guraya, S.S.: Comparative morphological and histochemical observations on the ovarian stromal com partment in mammals with special reference to steroidogenesis. Acta anat. 90: 250-284 (1974). Hammerstein, J.; Rice, B.F., and Savard, K.: Steroid hormone formation in the human ovary. I. Identifi cation of steroid formed in vitro from acetate-l-"C in the corpus luteum. J. clin. Endocr. Metab. 24: 597 605(1964). Levy, H.; Deane. H.W., and Rubin, B. L.: Visualiza tion of steroid-3/i-OL-dehydrogcnase activity in tissues of intact and hypophysectomizcd rat. Endo crinology 65: 932-943 (1959).
Lobel, B. L. and Levy, E.: Enzymic correlates of devel opment, secretory function and regression of fol licles and corpora lutea in the bovine ovary. Acta endocr., Copenh. 59: suppl. I (1968). Lucas, V.P.: Neufled. A.H.; Utterback. G .J.; Martin. P. A., and Stotz, E.: The effect of estrogen on the production of a peroxidase in the rat uterus. J. biol. Chcm. 214: 775-780 (1955). Rubin, L.B.: Deane, H.W.. and Hamilton, J.A.: Biochemical and histochemical identification of l'-3-/i-hydroxysteroid dehydrogenase activity in the adrenal cortex and ovaries of diverse mammals. Endocrinology 7.?: 748-763 (1963). Rubin, B.L.; Hilliard, J.: Hayward, J. H., and Deane, H. W .: Acute effects of gonadotropic hormones on rat and rabbit ovarian P-3/i-hydroxysteroid de hydrogenase activities. Steroids, suppl. I, p p .121130(1965). Saunders, B.C.; Holmes, S. A.G., and Stark, B. P.: Peroxidase (Butterworth. London 1964). Savard, K.; Marsh, J.M.. and Rice, B.F.: Gonado tropin and ovarian steroidogenesis. Recent Prog. Horm. Res. 21: 285-365 (1965). Taylor, F.B.: Histochemical changes in the normal and experimentally treated rats. Acta endocr.. Copenh. 36: 361-374(1961). Van Duija, P.: An improved histochemical benzidine blue peroxidase method and a note on the composi tion of the blue reaction product. Recueil 74: 771 777 (1951). Villee, C. A. and Engel, L. L.: Mechanism of action of steroid hormones. Mechanism of steroid hormones (London 1961). Wiest, W G. and Kidwell. W.R.: The regulation of progesterone secretion by ovarian dehydrogenase; in McKerns, The gonads, pp. 1-127 (Pergamon Press, London 1965). Zander. J.; Brendle, E.; Munstcrmann, A.M. von; Diczfalusy, E.; Martinsen, B., and Tillinger. K.G.: Identification and estimation of estradiol-17/f and estrone in human ovaries. Acta obstet. gynec. scand. 38: 724-736(1959).
Received: May 11, 1977 Prabha Agrawal, Department of Life Sciences, University of Indore, Indore (India)
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progesterone [Zander et al., 1959; Hammerstein et al., 1964; Savard et al., 1965]. The enzyme activities like zl“-3/f-OH-steroid de hydrogenases and 17/J-HSD in the mammals have been correlated with the synthesis of 2a-OH steroid and progesterone in the cells of the theca interna, interstitial gland cells and corpus luteum [Levy et al., 1959; Taylor, 1961: Rubin et al., 1963, 1965: Wiest and Kidwell, 1965], The secretory sites of progesterone in the normal and pregnant rats are the corpora lutea and the placenta, respectively. However, since peroxidase-mediated reactions are mani fold faster than dehydrogenase reactions, the association of high peroxidase activity in these regions and lack of activ ity in growing follicles and in IGT suggest that peroxidase may be involved in the luteal steroidogenesis as sug gested by us earlier [Agrawal and Laloraya, 1977],
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Histocheniical studies on the peroxidase localization in the rat ovary and uterus during various reproductive stages Prabha Agrawal and Mannwhan M . Laloraya Department of Life Sciences, University of Indore, Indore, India
Key words. Ovary • Rat • Peroxidase • Reproductive cycle • Pregnancy
Introduction The different components of the ovary, i.e. the follicle, corpus luteum and stroma, undergo complex morphological, histocheniical and biochemical changes during the reproductive cycle of the rat. After ovulation, the cells of the membrana granulosa show hypertrophy and become luteal cells which secrete massive amounts of progesterone. Progesterone is known to be a pre cursor of several other steroid hormones including estradiol that controls folliculogenesis. The corpora lutea of many mammals are the chief site of conversion of “ C-acetate to progesterone [Zander el at.. 1959; Hammerstein el a!., 1964; Savard el id.. 1965] and are known to be characterized by the presence of large quantities of sudanophilic lipids and the presence of the specific steroid dehydrogenases |Levy el al., 1959; Guraya. 1974], Steroidogenesis in the reproductive target organs has been related to the functioning of large number of specific dehydrogenases and isomerases [Villee and Engel. 1961], In the presence of specific dehydrogenases,
operative both during the development of the follicle in the membrana granulosa [Deane el al.. 1962; Rubin el al., 1963; Label and Levy, 1968] and in the derived corpus luteum, how the rate of oxidation reaction changes in such a manner so as to stimulate synthesis of progesterone at a faster rate is largely unknown. Recently, luteinizing hormone has been shown to in duce peroxidase in the corpora lutea accompanying the depletion of ascorbate in the rat ovary, and suggestion has been made that the free radical of ascorbate produced by the action of peroxidase on ascorbic acid may trigger oxidation of pregnenolone through a free radical mechanism, thus bringing about rapid forma tion of progesterone [Agrawal and Laloraya. 1977], The presence of peroxidase may, therefore, serve as a useful histocheniical indication of the process of lutcinization associated with intense vascularization of the ovary that occurs in response to luteinizing hormone. The present work reveals that not only the corpora lutea but also the uterus of the pregnant rats and the associated placenta, known to secrete large quantities of pro gesterone during pregnancy, are characterized by very high localization of peroxidase activity.
Downloaded by: King's College London 137.73.144.138 - 11/21/2017 6:43:11 PM
Abstract. A histocheniical study has been made of peroxidase changes in rat ovary and uterus during different developmental stages of the normal and pregnant rats. The peroxidase was found to be present in the corpus luteum of the ovary of both normal and pregnant rats as well as in the allantochorionic placenta, while the growing follicles in the immature and mature rat ovary showed no activity. The possible physiological significance of the peroxidase changes in relation to luteal steroidogenesis has been discussed.
Agra wal / Laloraya
95
Materials and methods
Observations The histochemical features of the ovary and uterus at different stages in normal and preg nant rats, especially in relation to the ocurrence of peroxidase and steroidogenesis, will be described here separately. Immature rat I-month-old rats show compact stroma tissue full of growing follicles and interstitial gland tissues (IGT). The granulosa cells of normal growing follicles are small. No activity of peroxidase could be seen either in the mem brana granulosa of the growing follicles or in the IGT (fig. I). The granular dark deposits seen in the stroma region and membrana gra nulosa are due to yellowish brown lipid drop lets which do not show peroxidase activity.
Fig. 1. A portion of a I-month-old rat ovary show ing no localization in the membrana granulosa (MG) and IGT.
Mature rat With the appearance of antrum filled with liquor folliculi, the granulosa and the sur rounding stromal elements begin to hyper trophy. In the final stages of follicular growth, the granulosa cells form a relatively thin layer, called membrana granulosa. The theca interna of the growing follicle consists of stromal ele ments which do not show much cytoplasmic differentiation. No peroxidase activity could be seen in the membrana granulosa while the theca interna cells of the atretic follicles show high peroxidase activity (fig. 4). As compared with the original granulosa cells, the luteal cells are hypertrophied glan dular tissue which is well vascularized. The most striking and significant change during the transformation of granulosa cells is the ap pearance of high peroxidase activity through out the cytoplasm of the latter. Ovaries from mature rats show heavily stained corpus
Downloaded by: King's College London 137.73.144.138 - 11/21/2017 6:43:11 PM
Colony-bred albino rats of different age groups of Wistar strain, maintained under laboratory conditions, were used in the present study. Mature rats showing regular 4- or 5-day cstrus cycles were sacrificed at estrus phase of the cycle which was checked by pre paring vaginal smears. Pregnancy, which lasted 21 days, was induced by mating with proven males, and only confirmed pregnant rats were taken for the study. Peroxidase was localized in freshly cut frozen sections (8-10 nm) of the ovary and uterus by the method of Van Dnija [1951] using benzidine as donor. The activity was also tested with two other donors, namely guaiacol and p-anisidine. The presence of peroxidase was indicated by the specifically colored products of the reaction: benzidine producing deep navy blue turning to dark brown, p-anisidine pink and guaiacol brown products. The specificity of the per oxidase system was checked by keeping controls with out H20 2, i.e. with donors alone, and by treating the sections for 30 min with different concentrations of KCN, namely 10 2 10 M potassium cyanide, before performing the lest.
Fig.2. A portion of preovulatory ovary of rat showing no activity of peroxidase in the growing follicles (GF). The membrana granulosa (MG) of a large growing follicle shows no activity of peroxidase, while the dense patches of IGT with no peroxidase activity are seen.
Fig.3. A portion of mature rat ovary showing localization of peroxidase in the corpus luteum (CL), while no activity could be seen in the membrana granulosa (MG) of the growing follicle.
Agrawal/Laloraya
Fig.4. A portion of a postovulatory 4-month-old rat ovary showing localization in the theca interna (TI) of atretic follicles and dotted localization (DL) in the stromal region. The black area seen in the IGT is due to heavy deposition of lipid material which does not give any reaction with any of the three donors in the peroxidase system.
Fig.5. A high-power view of corpus luteum (CL) from the mature ovary showing dense deposition of peroxidase in the corpus luteum.
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Fig. 6, 7. A portion o f the rat ovary at estrus showing intense navy blue localization of peroxidase in the corpora lutea (CL) and dotted localization (DL) in the stromal region. The IGT is seen with yellowbrown fat deposits showing no peroxidase activity. Fig. 8. A high-power view of corpus luteuni (CL) from the rat ovary at estrus showing dense navy blue deposition of peroxidase in the luteal cell mass (CL). No activity is seen in IGT containing yellow-brown fat deposits.
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Peroxidase changes during the reproductive cycle of rat
Fig. 9. A portion of postovulatory ovary showing diffuse localization of peroxidase in corpus albicans (CA).
Fig. 10. Cyanide-treated section of corpus luteum (CL) showing complete inhibition of peroxidase locali zation. Dotted localization (DL) is not affected by cyanide which is seen in the stromal region (S).
Agrawal/Laloraya
luteum (fig. 5, 6) with no activity in the mem brana granulosa of the growing follicles and IGT (fig. 2, 3, 6, 7). However, corpora lútea in ovary exhibited different intensities of per oxidase activity which appears to change with the regression of corpus luteum (fig. 9). The black patches observed in the IGT are again due to massive accumulation of yellowbrown lipid droplets devoid of any peroxidase activity. No activity was observed without H.,02 in the system and cyanide exerted a powerful inhibitory effect (fig. 10). The stromal elements, which are apparently derived from the theca interna and theca ex terna, are of small size. Apart from heavy localization in the corpus luteum, the stroma region shows sparsely distributed dotted cel lular localization (fig. 6). Cyanide treatment almost completely inhibited the localization in the corpus luteum. but the scattered dotted cellular localization was not much affected. Also the scattered dotted cellular localization was observed with another donor, namely, guaiacol. No localization could be obtained with p-anisidine as donor. These observations raise doubt about the presence of peroxidase activity in these cells. No activity of the per oxidase was seen in the uterus of the normal mature rat (fig. 12). The myometrium and se rous coat of the uterus show dotted cellular localization which was not inhibited by cya nide. The dotted localization observed in the stroma tissue of the ovary, in the myometrium and serous coat of the uterus, which is in sensitive to cyanide, appears to be due to the presence of some oxidizing substances which are known to produce the colored oxidation products nonenzymically. Pregnant rat During early pregnancy, well-vascularized luteal cells present in the ovary showed intense
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Fig. 11. Part of rat corpus lutcum (CL) in early stages of pregnancy showing intense activity of per oxidase in the luteal cell mass. Stroma (S) shows dotted localization.
Fig. 12. Uterus of normal rat showing only dotted localization (DL) in myometrium (M) and serous coat (SC). Note that no localization is seen in the endo metrium (H).
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Fig. 13. Uterus of pregnant rat showing heavy localization of peroxidase in epithelial cells of the endometrium (E) while dotted localization (DL) is seen in the stromal region (S). The myometrium (MM) shows no activity.
Fig. 14. A portion of pregnant rat uterus from the early half of gestation, showing navy blue deposition of peroxidase in the endometrium (E) while dotted localization is seen in the stroma (S) region. The myo metrium (M) shows no activity.
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Peroxidase changes during the reproductive cycle o f rat
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Agrawal/Laloraya
Thus, the growing follicles in the immature as well as the mature rats showed no perox idase activity, while the corpora lutea in the mature rats and of the pregnant as well as the allantochorionic placenta showed intense per oxidase activity.
Fig. 15. A high-powcr view of early embryo (EY) in the uterus. The allantochorionic placenta (ACP) shows intense activity of peroxidase. The black patches ob served in the embryo a re. due to its compact nature and yellow-brown color which was devoid of peroxidase activity.
peroxidase activity (fig. 8, 11 ). The intensity of the localization was much higher as compared to the corpora lutea of normal rats at estrus. The uteri of pregnant rats show a welldeveloped decidua exhibiting extremely high peroxidase activity (fig. 13, 14). No activity could be seen in the myometrium and serous coat of the uterus at this stage of pregnancy. The entire length of the decidua was deeply stained, the endometrium lining exhibiting maximum localization. Some dotted cellular localization is seen in the stroma region of the decidua. The attachment between the embryo and the decidua, i.e. allantochorionic placenta, which develops during the later stages of preg nancy, showed intense localization of per oxidase (fig. 15).
The presence of peroxidase in the spleen, stomach, adrenal glands, eosinophils, intestine and liver has been reported [Saunders et at., 1964], Lucas et at. [1955] have shown the pres ence of peroxidase in the uterus while it under goes rapid proliferation during the different phases of the cycle, and this activity is even more pronounced in the estrogen-stimulated uterus of the ovariectomized rat. These reports of the occurrence of peroxidase in the genital organs, however, did not specify the localiza tion and its possible role in luteal steroidogen esis. Guraya [1974], on the basis of presence of sudanophilic granules and its changes under gonadotrophic hormone action, has suggested the possibility of interstitial gland cells acting as sites for steroidogenesis. These sites are well known to be associated with storage of lipid material including phospholipids and triglycerides. The observation that cholesterol could be demonstrated in interstitial gland cells and under conditions of lowered progesterone secretion and not during active secretion might suggest that the mobilization of the precursors for the biosynthesis of progesterone is con trolled by the corpus luteum. Since no activity of peroxidase could be demonstrated in inter stitial gland cells it appears to be an unlikely site for luteal steroidogenesis. The corpora lutea of many mammals are the chief site of conversion of "C-acetate to
Downloaded by: King's College London 137.73.144.138 - 11/21/2017 6:43:11 PM
Discussion
Peroxidase changes during ihe reproductive cycle of rat
References Agrawal, P. and Laloraya, M.M.: Induction of per oxidase in corpora lutea of rat ovary by luteinizing hormone. Biochem. J. 166: 205 208 (1977) Deane. H .W .; Lobel, B.L., and Romney, S. L.: En zymic histochemistry of normal human ovaries of the menstrual cycle, pregnancy and the early puerperium. Am. J. Obstet. Gynec. 83: 281-294 (1962). Guraya, S.S.: Comparative morphological and histochemical observations on the ovarian stromal com partment in mammals with special reference to steroidogenesis. Acta anat. 90: 250-284 (1974). Hammerstein, J.; Rice, B.F., and Savard, K.: Steroid hormone formation in the human ovary. I. Identifi cation of steroid formed in vitro from acetate-l-"C in the corpus luteum. J. clin. Endocr. Metab. 24: 597 605(1964). Levy, H.; Deane. H.W., and Rubin, B. L.: Visualiza tion of steroid-3/i-OL-dehydrogcnase activity in tissues of intact and hypophysectomizcd rat. Endo crinology 65: 932-943 (1959).
Lobel, B. L. and Levy, E.: Enzymic correlates of devel opment, secretory function and regression of fol licles and corpora lutea in the bovine ovary. Acta endocr., Copenh. 59: suppl. I (1968). Lucas, V.P.: Neufled. A.H.; Utterback. G .J.; Martin. P. A., and Stotz, E.: The effect of estrogen on the production of a peroxidase in the rat uterus. J. biol. Chcm. 214: 775-780 (1955). Rubin, L.B.: Deane, H.W.. and Hamilton, J.A.: Biochemical and histochemical identification of l'-3-/i-hydroxysteroid dehydrogenase activity in the adrenal cortex and ovaries of diverse mammals. Endocrinology 7.?: 748-763 (1963). Rubin, B.L.; Hilliard, J.: Hayward, J. H., and Deane, H. W .: Acute effects of gonadotropic hormones on rat and rabbit ovarian P-3/i-hydroxysteroid de hydrogenase activities. Steroids, suppl. I, p p .121130(1965). Saunders, B.C.; Holmes, S. A.G., and Stark, B. P.: Peroxidase (Butterworth. London 1964). Savard, K.; Marsh, J.M.. and Rice, B.F.: Gonado tropin and ovarian steroidogenesis. Recent Prog. Horm. Res. 21: 285-365 (1965). Taylor, F.B.: Histochemical changes in the normal and experimentally treated rats. Acta endocr.. Copenh. 36: 361-374(1961). Van Duija, P.: An improved histochemical benzidine blue peroxidase method and a note on the composi tion of the blue reaction product. Recueil 74: 771 777 (1951). Villee, C. A. and Engel, L. L.: Mechanism of action of steroid hormones. Mechanism of steroid hormones (London 1961). Wiest, W G. and Kidwell. W.R.: The regulation of progesterone secretion by ovarian dehydrogenase; in McKerns, The gonads, pp. 1-127 (Pergamon Press, London 1965). Zander. J.; Brendle, E.; Munstcrmann, A.M. von; Diczfalusy, E.; Martinsen, B., and Tillinger. K.G.: Identification and estimation of estradiol-17/f and estrone in human ovaries. Acta obstet. gynec. scand. 38: 724-736(1959).
Received: May 11, 1977 Prabha Agrawal, Department of Life Sciences, University of Indore, Indore (India)
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progesterone [Zander et al., 1959; Hammerstein et al., 1964; Savard et al., 1965]. The enzyme activities like zl“-3/f-OH-steroid de hydrogenases and 17/J-HSD in the mammals have been correlated with the synthesis of 2a-OH steroid and progesterone in the cells of the theca interna, interstitial gland cells and corpus luteum [Levy et al., 1959; Taylor, 1961: Rubin et al., 1963, 1965: Wiest and Kidwell, 1965], The secretory sites of progesterone in the normal and pregnant rats are the corpora lutea and the placenta, respectively. However, since peroxidase-mediated reactions are mani fold faster than dehydrogenase reactions, the association of high peroxidase activity in these regions and lack of activ ity in growing follicles and in IGT suggest that peroxidase may be involved in the luteal steroidogenesis as sug gested by us earlier [Agrawal and Laloraya, 1977],
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